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Retrospective studies of breast cancer patients suggest that primary tumor Her-2 overexpression or trastuzumab therapy is associated with a devastating complication: the development of central nervous system (brain) metastases. Herein, we present Her-2 expression trends from resected human brain metastases and data from an experimental brain metastasis assay, both indicative of a functional contribution of Her-2 to brain metastatic colonization. Of 124 archival resected brain metastases from breast cancer patients, 36.2% overexpressed Her-2, indicating an enrichment in the frequency of tumor Her-2 overexpression at this metastatic site. Using quantitative real-time PCR of laser capture microdissected epithelial cells, Her-2 and epidermal growth factor receptor (EGFR) mRNA levels in a cohort of 12 frozen brain metastases were increased up to 5- and 9-fold, respectively, over those of Her-2–amplified primary tumors. Co-overexpression of Her-2 and EGFR was also observed in a subset of brain metastases. We then tested the hypothesis that overexpression of Her-2 increases the colonization of breast cancer cells in the brain in vivo. A subclone of MDA-MB-231 human breast carcinoma cells that selectively metastasizes to brain (231-BR) overexpressed EGFR; 231-BR cells were transfected with low (4- to 8-fold) or high (22- to 28-fold) levels of Her-2. In vivo, in a model of brain metastasis, low or high Her-2–overexpressing 231-BR clones produced comparable numbers of micrometastases in the brain as control transfectants; however, the Her-2 transfectants yielded 3-fold greater large metastases (>50 μm2; P < 0.001). Our data indicate that Her-2 overexpression increases the outgrowth of metastatic tumor cells in the brain in this model system. [Cancer Res 2007;67(9):4190–8]

Cancer metastasis is a significant contributor to breast cancer patient morbidity and mortality. In order to develop new anti-metastatic therapies, we need to understand the biological and biochemical mechanisms of metastasis. Toward these efforts, we and others have studied metastasis suppressor genes, which halt metastasis in vivo without affecting primary tumor growth. The first metastasis suppressor gene identified was nm23, also known as NDP kinase. Nm23 represents the most widely validated metastasis suppressor gene, based on transfection and knock-out mouse strategies. The biochemical mechanism of metastasis suppression via Nm23 is unknown and likely complex. Two potential mechanisms include binding proteins and a histidine kinase activity. Elevation of Nm23 expression in micrometastatic tumor cells may constitute a translational strategy for the limitation of metastatic colonization in high risk cancer patients. To date, medroxyprogesterone acetate (MPA) has been identified as a candidate compound for clinical testing.

Background: Reestablishment of metastasis suppressor gene expression may constitute a therapeutic strategy for high-risk breast cancer patients. We previously showed that medroxyprogesterone acetate (MPA), a progestin that has been tested as treatment for advanced breast cancer, elevates expression of the Nm23-H1 metastasis suppressor gene in hormone receptor–negative metastatic human breast carcinoma cell lines in vitro via a glucocorticoid receptor–based mechanism. Here, we tested whether MPA treatment inhibits metastatic colonization of a hormone receptor–negative breast cancer cell line in vivo. Methods: We tested the soft-agar colony-forming efficiency of untransfected MDA-MB-231T human breast carcinoma cells and MDA-MB-231T cells transfected with antisense Nm23-H1 in the presence and absence of MPA. Pharmacokinetic studies were used to establish dose and injection schedules that led to MPA serum levels in mice similar to those achievable in humans. For in vivo studies, nude mice were injected intravenously with MDA-MB-231T cells. After 4 weeks, mice were randomized to control or MPA arms. Endpoints included incidence, number, and size of gross pulmonary metastases; Nm23-H1 protein expression in gross metastases; and side effects. All statistical tests were two-sided. Results: MPA reduced colony formation of MDA-MB-231T cells by 40%–50% but had no effect on colony formation of Nm23-H1 antisense transfectants. Metastases developed in 100% (95% confidence interval [CI] = 78% to 100% and 77% to 100%, respectively) of control mice injected with MDA-MB-231T cells. In two independent experiments, only 73% (95% CI = 45% to 92%) and 64% (95% CI = 35% to 87%) of mice injected with 2 mg of MPA developed metastases. Mice injected with 2 mg of MPA showed reductions in the mean numbers, per mouse, of all metastases and of large (>3 mm) metastases ( P = .04 and .013, respectively). Nm23-H1 was expressed at high levels in 43% of pulmonary metastases in MPA-treated mice but only 13% of metastases in untreated mice. Mice receiving at least 1-mg doses of MPA gained more weight than control-treated mice but exhibited no bone density alterations or abnormal mammary fat pad histology. Conclusion: Our preclinical results show that MPA appears to elevate Nm23-H1 metastasis suppressor gene expression, thereby reducing metastatic colonization. The data suggest a new use for an old agent in a molecularly defined subset of breast cancer patients.

Metastatic disease is a significant contributor to cancer patient mortality. We previously reported that the Kinase Suppressor of Ras1 (KSR1) scaffold protein for the Erk mitogen-activated protein kinase pathway coimmunoprecipitated the metastasis suppressor protein Nm23-H1. We now hypothesize that altered expression levels of Nm23-H1 influence the binding properties, stability, and function of the KSR1 scaffold. Increased coimmunoprecipitation of Hsp90 with KSR1 was observed in either stable or transient transfectants of nm23-H1 in MDA-MB-435 human breast carcinoma cells. Similar trends were also observed in the cytoplasmic and nuclear fractions of cells. Cells expressing high levels of Nm23-H1 exhibited increased KSR1 degradation in the presence of either cycloheximide or an Hsp90-directed drug currently in clinical trial, 17-allylamino-17-demethoxygeldanamycin (17-AAG). In agreement with KSR1 degradation data, high-Nm23-H1-expression cells were preferentially inhibited in anchorage-independent colonization assays by 17-AAG. KSR1 scaffold binding patterns are dynamic in both the cytoplasmic and nuclear compartments, modulated by metastasis suppressor expression. Metastasis suppressor expression levels can impact traditional signaling pathways, such as the Erk pathway, resulting in altered tumor cell sensitivity to cancer therapeutics.

Metastatic disease remains a significant contributor to morbidity and mortality in patients with breast cancer. An improved molecular and biochemical understanding of the metastatic process is expected to fuel the development of new therapeutic approaches. The suppression of tumor metastasis, despite tumor cell expression of oncogenes and metastasis-promoting events, has become a diverse and fruitful field of investigation. Although many genetic events promote metastasis, several genes show relatively reduced expression levels in metastatic tumor cells in mouse model systems and in aggressive human tumors. Re-expression of a metastasis-suppressor gene in a metastatic tumor cell line results in a significant reduction in metastatic behavior in vivo with no effect on tumorigenicity. The known metastasis-suppressor gene products nm23, KAI1, mitogen-activated protein kinase kinase 4, breast cancer metastasis suppressor-1, KiSS1, RHOGDI2, CRSP3, and vitamin D3-upregulated protein/thioredoxin interacting protein exhibit unexpected biochemical functions that have shed new light on signaling events that are important in metastasis. Most metastasis suppressors function at the translationally important stage of outgrowth of micrometastatic tumor cells at a distant site. We hypothesize that elevation of metastasis suppressor gene expression in micrometastatic tumor cells in the adjuvant high-risk population of patients with breast cancer will halt metastatic colonization and have a clinical benefit. DNA methylation inhibitors have shown limited promise in increasing metastasis-suppressor gene expression, and ligands of the nuclear hormone receptor family are currently under investigation in vitro and in vivo. Clinical testing of agents that increase metastasis-suppressor gene expression is expected to require tailored trial designs.

CD40 is present on both normal and neoplastic B-lineage cells. CD40 stimulation of normal B cells has been shown to promote normal growth and differentiation, whereas aggressive histology B lymphomas are growth inhibited. The inhibition of neoplastic B-cell growth is believed to occur via activation-induced cell death in which stimuli that typically promote the growth of normal cells prevent the growth of their neoplastic counterparts. We show here that CD40 stimulation using either a soluble recombinant human CD40 ligand (srhCD40L) or anti-CD40 monoclonal antibody resulted in apoptosis of human Burkitt lymphoma cell lines. Additional studies examining the mechanism of CD40-mediated death revealed an increase in bax messenger RNA with a subsequent increase in Bax protein in the mitochondria of the treated cells. In vitro exposure of the cells to bax antisense oligonucleotides resulted in a significant decline in Bax protein levels and partial protection from CD40-mediated death, indicating that induction of Bax was at least one mechanism underlying this inhibitory effect of CD40 stimulation on lymphomas. When immunodeficient mice bearing Burkitt lymphoma were treated with srhCD40L, significant increases in survival were observed indicating a direct antitumor effect as a result of CD40 stimulation in vivo. Overall, these results demonstrate that CD40 ligation of aggressive histology B-lymphoma cells results in inhibition both in vitro and in vivo and thus may be of potential clinical use in their treatment.

Long-term elevation of metastasis suppressor gene expression in micrometastases represents a novel therapeutic strategy for breast and other cancers. We searched for well-tolerated compounds that could elevate Nm23 metastasis suppressor expression in metastatic human breast cancer cell lines.MDA-MB-435 and MDA-MB-231 human breast carcinoma cells were treated with dexamethasone or medroxyprogesterone acetate (MPA) in cultures containing either charcoal-stripped serum or FCS. Aspects of nm23 expression and function were determined.Previous investigation of the nm23-H1 promoter suggested that glucocorticoids may contribute to the elevation of Nm23-H1 expression. Dexamethasone elevated Nm23-H1 and Nm23-H2 protein levels in two metastatic human breast carcinoma cell lines 2-3-fold over a 4-day time course when cultured in steroid-free culture medium, with high-dose inhibition, via a traditional transcriptional mechanism. Elevation of Nm23-H1 expression was not observed using FCS-containing culture medium, which contains endogenous levels of corticosteroids, limiting the potential in vivo use of dexamethasone. MPA was investigated as a glucocorticoid receptor agonist. MPA elevated breast carcinoma Nm23-H1 protein expression 3-fold over a 10 nM to 1 micro M dose range when cultured in steroid-free or FCS-containing medium, with a shorter time course. Elevation of Nm23-H1 expression in the presence of endogenous corticosteroids found in FCS involved a distinct, glucocorticoid receptor-dependent, posttranscriptional mechanism of action. MPA had no effect on proliferation in vitro but reduced the soft agar colonization of metastatic breast cancer cell lines by approximately 50%.MPA represents a first generation lead agent for the elevation of Nm23-H1 metastasis suppressor expression and the inhibition of metastatic colonization.